a) Field of the Invention
The invention is directed to a method and an arrangement in fluorescence microscopy, particularly laser scanning microscopy, fluorescence correlation spectroscopy, and nearfield scanning microscopy, for examination of predominantly biological specimens, preparations and associated components. This includes methods for screening active ingredients based on fluorescence detection (high throughput screening). The transition from the detection of a few broad-spectrum dye bands to the simultaneous acquisition of whole spectra opens up new possibilities for the identification, separation and allocation of mostly analytic or functional specimen characteristics to spatial partial structures or dynamic processes. Therefore, simultaneous examination of specimens with multiple fluorophores with overlapping fluorescence spectra are even possible in spatial structures of thick specimens. In addition, it is possible to detect local spectral shifts of emission bands of the dyes and to allocate them to the spatial structures. The data acquisition rate is not reduced by the arrangement.
b) Description of the Related Art
A typical area of application of light microscopy for examining biological preparations is fluorescence microscopy (Pawley, “Handbook of Biological Confocal Microscopy”; Plenum Press 1995). In this case, determined dyes are used for specific marking of cell parts.
The irradiated photons having a determined energy excite the dye molecules, through the absorption of a photon, from the ground state to an excited state. This excitation is usually referred to as one-photon or single-photon absorption (FIG. 1a). The dye molecules excited in this way can return to the ground state in various ways. In fluorescence microscopy, the most important is the transition with emission of a fluorescence photon. Because of the Stokes shift, there is generally a red shift in the wavelength of the emitted photon in comparison to the excitation radiation; that is, it has a greater wavelength. Stokes shift makes it possible to separate the fluorescence radiation from the excitation radiation.
The fluorescent light is split off from the excitation radiation by suitable dichroic beam splitters in combination with blocking filters and is observed separately. This makes it possible to show individual cell parts that are dyed with different dyes. In principle, however, several parts of a preparation can also be dyed simultaneously with different dyes which bind in a specific manner (multiple fluorescence). Special dichroic beam splitters are used again to distinguish the fluorescence signals emitted by the individual dyes.
In addition to excitation of dye molecules with a high-energy photon (single-photon absorption), excitation with a plurality of low-energy photons is also possible (FIG. 1b). The sum of energies of the single photons corresponds approximately to a multiple of the high-energy photon. This type of excitation of dyes is known as multi-photon absorption (Corle, Kino, “Confocal Scanning, Optical Microscopy and Related Imaging Systems”; Academic Press 1996). However, the dye emission is not influenced by this type of excitation, i.e., the emission spectrum undergoes a negative Stokes shift in multi-photon absorption; that is, it has a smaller wavelength compared to the excitation radiation. The separation of the excitation radiation from the emission radiation is carried out in the same way as in single-photon excitation.
The prior art will be explained more fully in the following by way of example with reference to a confocal laser scanning microscope (LSM) (FIG. 2[L1].
An LSM is essentially composed of four modules: light source, scan module, detection unit and microscope. These modules are described more fully in the following. In addition, reference is had to DE19702753A1.
Lasers with different wavelengths are used in an LSM for specific excitation of different dyes in a preparation. The choice of excitation wavelengths is governed by the absorption characteristics of the dyes to be examined. The excitation radiation is generated in the light source module. Various lasers (argon, argon/krypton, Ti:Sa lasers) are used for this purpose. Further, the selection of wavelengths and the adjustment of the intensity of the required excitation wavelength is carried out in the light source module, e.g., using an acousto-optic crystal. The laser radiation subsequently reaches the scan module via a fiber or a suitable mirror arrangement.
The laser radiation generated in the light source is focussed in the preparation in a diffraction-limited manner by means of the objective (2) via the scanner, scanning optics and tube lens. The focus scans the specimen in a point raster in x-y direction. The pixel dwell times when scanning over the specimen are mostly in the range of less than one microsecond to several seconds.
In confocal detection (descanned detection) of fluorescent light, the light emitted from the focal plane (specimen) and from the planes located above and below the latter reaches a dichroic beam splitter (MDB) via the scanner. This dichroic beam splitter separates the fluorescent light from the excitation light. The fluorescent light is subsequently focused on a diaphragm (confocal diaphragm/pinhole) located precisely in a plane conjugate to the focal plane. In this way, fluorescent light components outside of the focus are suppressed. The optical resolution of the microscope can be adjusted by varying the size of the diaphragm. Another dichroic blocking filter (EF) which again suppresses the excitation radiation is located behind the diaphragm. After passing the blocking filter, the fluorescent light is measured by means of a point detector (PMT).
When using multi-photon absorption, the excitation of the dye fluorescence is carried out in a small volume at which the excitation intensity is particularly high. This area is only negligibly larger than the detected area when using a confocal arrangement. Accordingly, a confocal diaphragm can be dispensed with and detection can be carried out directly following the objective (non-descanned detection).
In another arrangement for detecting a dye fluorescence excited by multi-photon absorption, descanned detection is carried out again, but this time the pupil of the objective is imaged in the detection unit (nonconfocal descanned detection).
From a three-dimensionally illuminated image, only the plane (optical section or slice) located in the focal plane of the objective is reproduced by the two detection arrangements in connection with corresponding single-photon absorption or multi-photon absorption. By recording or plotting a plurality of optical slices in the x-y plane at different depths z of the specimen, a three-dimensional image of the specimen can be generated subsequently in computer-assisted manner.
Accordingly, the LSM is suitable for examination of thick preparations. The excitation wavelengths are determined by the utilized dye with its specific absorption characteristics. Dichroic filters adapted to the emission characteristics of the dye ensure that only the fluorescent light emitted by the respective dye will be measured by the point detector.
Currently, in biomedical applications, a number of different cell regions are labeled simultaneously by different dyes (multifluorescence). In the prior art, the individual dyes can be detected separately based on different absorption characteristics or emission characteristics (spectra). For this purpose, an additional splitting of the fluorescent light of a plurality of dyes is carried out with the secondary beam splitters (DBS) and a separate detection of the individual dye emissions is carried out in separate point detectors (PMT x). With the arrangement described above, it is impossible for the user to flexibly adapt detection and excitation to corresponding new dye characteristics. Instead, new dichroic beam splitters and blocking filters must be created for every (new) dye. In an arrangement according to DE . . . , the fluorescent light is split spectrally by means of a prism. The method differs from the above-described arrangement with dichroic filters only in that the characteristic of the utilized filter is adjustable. However, it is still preferable to record the emission band of a dye by point detector.
The limits of the previous detection devices are reached when the emission spectra of two dyes overlap. In order to prevent crosstalk between two dyes, the spectral detection area must be limited. In this case, the area in which the two dyes overlap is simply cut out and not detected. This reduces the efficiency of the detection unit. The same signal-to-noise ratio can be achieved only by increasing the excitation output, which could lead to damage to the preparation. Therefore, at the present time, a maximum of six different dye probes are used simultaneously, since the dyes could not otherwise be separated because of the heavily overlapping emission bands.
Previously, dyes were modified in such a way that they differed from each other either with respect to their absorption characteristics or their emission characteristics. FIG. 3a shows the emission spectra of different typical dyes. The emission signal is plotted over wavelength. It will be noted that the dyes designated by 1 to 4 differ from one another in the position and shape of their emission spectra. In most cases, however, these dyes are toxic for in vivo preparations. Therefore, investigations of the evolution of cell bonds in living preparations are not possible.
Naturally occurring dyes, so-called fluorescent proteins (GFP, YFP, CFP, TOPAS, GFT, RFP), were discovered in the late 1990s (Clonetech, USA). These dyes are distinguished by their reduced influence on specimens. They are therefore particularly suitable for labeling cell regions in living preparations. However, it is disadvantageous that the dyes differ only slightly with respect to their emission characteristics. FIG. 3b shows the emission signals as a function of the wavelength for the dyes GFP, Topas, GFT and Cyan-FP.
With conventional methods, only CFP and RFP, due to their altered absorption characteristics, could be separated from the rest efficiently, i.e., in sequential image recording. It is not possible to separate the dyes GFP and GFT at all by conventional means.
In another method for determining the localization of two proteins, both proteins are labeled with different dyes, wherein the emission spectrum of the first dye overlaps with the absorption spectrum of the second dye. The first dye is subsequently excited to fluorescence with a suitable wavelength. When both molecules are located very close to one another (<10 nm), the emission radiation of the first dye can be absorbed by the second, so that the second dye and not the first dye subsequently emits. FIG. 3d shows the energy level diagram for this process which is known in the literature as Fluorescent Resonant Energy Transfer (FRET) (Fan, et al., Biophysical Journal, V 76, May 1999, P 2412-2420). When the emission radiation of the two dyes is detected with this method and the ratio of both detection signals is determined, the distance between the two molecules can be determined.
Further, it is known that the emission spectrum of a dye found in a biological preparation can differ from the emission spectrum measured in a dye cuvette. FIG. 3c shows the emission spectra of a dye as a function of the environment in which the dye is found. In the Figure, the emission signal is plotted over wavelength.
The wavelength shift can amount to several times 10 nm. More precise investigations of the dependency of this wavelength shift on the environment were not known previously, since an investigation of this kind was very difficult to carry out using the methods according to the prior art. While spectrometers are also currently used in combination with an LSM, a conventional, usually high-resolution spectrometer is used instead of a point detector (Patent: Dixon, et al. U.S. Pat. No. 5,192,980). However, these spectrometers can record an emission spectrum only point by point or as an average over a region. Thus, this is a type of spectroscopy. In another arrangement, the lifetime of the dye fluorescence is measured, so that the type of environment can be deduced. However, a long data acquisition time would be required for recording a complete image. Therefore, these methods can only be used conditionally for examining living preparations.
In another application of fluorescence microscopy, the ion concentration (e.g., Ca+, K+, Mg2+, ZN+, . . . ) is determined, particularly in biological preparations. Special dyes or dye combinations (e.g., Fura, Indo, Fluo; Molecular Probes, Inc.) having a spectral shift depending on the ion concentration are used for this purpose. FIG. 4a shows the emission spectra of Indo-1 in dependence on the concentration of calcium ions. FIG. 4b shows an example of the emission spectra depending on the calcium ion concentration using the combination of Fluo-3 and Fura Red dyes. These special dyes are known as emission ratio dyes. When the two fluorescence areas shown in FIG. 4a are detected again and the ratio of both intensities is taken, the corresponding ion concentration can be determined. In these measurements, the examination is usually directed to dynamic change in the ion concentration in living preparations requiring a time resolution of less than one millisecond.